Little is known about the mechanisms or pathophysiology responsible for age-related decline of internal anal sphincter (IAS) function. Decreased mechanical efficiency of smooth muscle of the IAS results in decreased closure pressure of the sphincter, thus greatly contributing to fecal incontinence which is disproportionately prevalent in the elderly. Recent advances in tissue engineering provide us with an excellent in vitro model mimicking in vivo function to study the effects of aging on the molecular mechanisms of the IAS smooth muscle. We have for the first time, bioengineered three- dimensional (3-D) rings from isolated smooth muscle cells from human IAS. These rings developed tone, responded to acetylcholine in a dose-dependent manner and relaxed upon the exogenous addition of the relaxant mediator 8-Br-cAMP. Preliminary results from Human IAS and Human circular colonic smooth muscle cells (CSMC) indicate: 1) Sequestration of RhoA, phospho-PKC1 (S657) and HSP27 only in the caveolin-rich lipid raft microdomains of IAS cells at rest and not CSMC;2) a greater expression of HSP27, RhoA, PKC1 and phospho CPI-17 in Human IAS cells vs. CSMC. Rings from old Rat IAS cells showed decreased contractile response (maximal force generation 5N and time-to-peak response) when compared to IAS rings from adult rats that correlated with decreased HSP27 phosphorylation. Reduced phosphorylation of HSP27 affects actin cytoskeleton stability leading to disturbed caveolae formation. Overexpression of phosphomimic-HSP27 in old IAS smooth muscle cells exhibited increased association of PKC1 and HSP27 with caveolin-1, and also reinstated the magnitude of force generated and the time-to-peak contraction in IAS rings bioengineered from these cells. The specific aims of this grant proposal are: 1) Develop a 3-D physiological model of the IAS bioengineered in vitro from cells isolated from the IAS of human and of adult and aged rats, and examine the effect of aging on the molecular mechanisms of tonic IAS smooth muscle contraction 2) Study the role of phosphorylated-HSP27 in age-related decline of IAS smooth muscle function, and 3) Examine the reinstatement of physiological contractile function in 3-D IAS rings bioengineered from IAS smooth muscle cells transfected with phosphomimic-HSP27 cDNA.